Apparatuses and methods for detecting molecules and binding energy

ABSTRACT

The present disclosure provides apparatuses and methods for analyzing the presence of charged analytes and/or the binding force between charged analytes and a capture probe. The apparatuses and methods of the present disclosure can be operated in a multiplexed format to perform various assays of clinical significance for example.

CROSS-REFERENCE

This application is a Continuation of U.S. application Ser. No.15/673,653, filed Aug. 10, 2017, which is a Continuation of ApplicationNo. PCT Serial No. PCT/US2016/017181, filed Feb. 9, 2016, which claimsthe benefit of U.S. Provisional Patent Application Ser. No. 62/114,474,filed Feb. 10, 2015, each of which is incorporated herein by referencein its entirety.

BACKGROUND

Various types of molecules can recognize other molecules by theformation of multiple (numbers and/or types of) non-covalent bonds(e.g., van der Waals, hydrogen bonds, coulombic attractions andhydrophobic bonds), which can be combined in a particular spatialorientation. These molecular recognition interactions can be reversibleand of moderate to high specificity. Examples of such molecularinteractions include antibody-antigen interactions and nucleic acidhybridizations. Such interactions are important for biology and severalmature and developing industries, such as medical diagnostics,therapeutics, biotechnology, agriculture, fuel and chemical production,defense, environmental monitoring, food and food safety to name a few.

SUMMARY

Recognized herein is the need for new and improved sensors and methodsfor detecting the presence of molecular interactions and for measuringthe force associated with molecular interactions. The present disclosureprovides such sensors and methods for using the sensors. Withoutlimitation, the apparatus and methods of the present disclosure haveadvantages in differentiating between specific (e.g., strong) andnon-specific (e.g., weak) molecular interactions and therefore, candistinguish between false positive and true positive signals. Thepresent apparatuses and methods may be suitable for multiplexing (e.g.,performing 5, 10, 10², 10³, 10⁴, 10⁵, 10⁶ or more measurements inparallel) and directly using complex mixtures having two or moreanalytes such as biological fluids (e.g., blood) or environmentalsamples. In some embodiments, the apparatuses and methods describedherein can be used to measure the force associated with molecularinteractions, spanning multiple orders of magnitude in strength (e.g.,between about 1 piconewton (pN) and about 400 pN).

In an aspect, a method for detecting a presence of a target analytecomprises (a) activating at least one sensor comprising one or moresurfaces between two or more electrodes, wherein the one or moresurfaces comprise two or more immobilized capture probes; (b) bringingthe one or more surfaces in contact with a solution containing orsuspected of containing a target analyte and a non-target analyte, underconditions sufficient to permit the target analyte and non-targetanalyte to each bind to a given one of the two or more immobilizedcapture probes; (c) applying a voltage across the two or more electrodesthat is sufficient to release the non-target analyte, but not the targetanalyte, from a given one of the two or more immobilized capture probes;and (d) detecting a signal indicative of a presence and/or bindingenergy of the target analyte on the one or more surfaces. In someembodiments, the target analyte and/or the non-target analyte is acharged analyte. In some embodiments, the voltage is sufficient to exertan applied force of at least about 1 piconewton (pN) on the non-targetanalyte. In some embodiments, the applied force is at least about 10 pN.In some embodiments, the voltage is sufficient to generate an electricfield having a strength of at least about 10³ volts per meter.

In some embodiments, the method further comprises applying an additionalvoltage across the two or more electrodes that is sufficient to releasethe target analyte from a given one of the two or more immobilizedcapture probes. In some embodiments, the additional voltage issufficient to exert an applied force of at least about 1 pN on thetarget analyte. In some embodiments, the applied force is at least about10 pN. In some embodiments, the method further comprises detecting thepresence of the target analyte on the one or more surfaces subsequent toapplication of the additional voltage. In some embodiments, the voltageis less than the additional voltage. In some embodiments, the voltageand the additional voltage are individual voltages of a continuouslyapplied voltage that is changed over time. In some embodiments, theadditional voltage is applied in the absence of washing the one or moresurfaces.

In some embodiments, the applied voltage is sufficient to generate anelectric field having a strength of less than about 10⁹ volts per meter(V/m) and the additional applied voltage is sufficient to generate anadditional electric field having a strength of less than about 10⁹ V/m.

In some embodiments, each of the target analyte and non-target analytenon-covalently binds to a given one of the two or more immobilizedcapture probes to form a target probe-analyte complex immobilized and anon-target probe-analyte complex immobilized on the one or moresurfaces, respectively.

In some embodiments, the applied voltage is increased from a firstvoltage to a second voltage over time. In some embodiments, the appliedvoltage is increased from the first voltage to the second voltage at arate of at least about 1 millivolt per second. In some embodiments, theapplied voltage is increased from the first voltage to the secondvoltage over a period of time greater than about 10 microseconds. Insome embodiments, the applied voltage is increased from the firstvoltage to the second voltage over a period of time less than about 1second. In some embodiments, the applied voltage is a periodic voltagewaveform. In some embodiments, the periodic voltage waveform has afrequency of less than about 1 gigahertz. In some embodiments, theperiodic voltage waveform has a frequency of greater than about 1millihertz.

In some embodiments, the method further comprises monitoring a signalindicative of the presence of the target analyte on the one or moresurfaces as the applied voltage is varied over time. In someembodiments, the method further comprises determining a binding force ofthe target analyte bound to a given one of the two or more immobilizedcapture probes.

In some embodiments, the applied force is less than a binding force ofthe target analyte bound to a given one of the two or more immobilizedcapture probes.

In some embodiments, the solution comprises charged molecules that bindwith the target analyte and/or the non-target analyte, to provide achange in charge on the target analyte and/or non-target analyte. Insome embodiments, the charged molecules are antibodies. In someembodiments, (c) and/or (d) is performed in the absence of washing theone or more surfaces.

In some embodiments, the presence of the target analyte on the one ormore surfaces is detected by measuring a signal associated with thetarget analyte. In some embodiments, the signal is a charge signal. Insome embodiments, the charge signal is detected using a sensingelectrode and a reference electrode. In some embodiments, the chargesignal is a voltage. In some embodiments, the signal indicative of thepresence and/or binding energy of the target analyte on the one or moresurfaces is detected by measuring an optical signal associated with thetarget analyte. In some embodiments, the optical signal is afluorescence signal. In some embodiments, the fluorescence signal isprovided by a fluorescent probe covalently or non-covalently attached tothe target analyte. In some embodiments, the signal indicative of thepresence and/or binding energy of the target analyte on the one or moresurfaces is detected using surface plasmon resonance.

In some embodiments, the one or more surfaces is substantially planar.In some embodiments, the one or more surfaces comprise an electricallyinsulating layer and an electrically conducting layer. In someembodiments, the electrically conducting layer is in communication withthe solution. In some embodiments, the electrically conducting layer iselectrically isolated from the solution and the electrically conductinglayer is electrically biased. In some embodiments, the electricallyconducting layer comprises platinum. In some embodiments, the one ormore surfaces comprise a membrane having a thickness of between about0.08 nanometers and 1 millimeter. In some embodiments, the one or moresurfaces comprise a field confining feature that concentrates anelectric field to a strength of at least about 10³ volts per meter. Insome embodiments, the field confining feature comprises an orifice inthe one or more surfaces, which one or more surfaces comprise anelectrically insulating layer and an electrically conducting layer.

In some embodiments, the two or more immobilized capture probes arenucleic acid molecules that hybridize with the target analyte. In someembodiments, the target analyte is a nucleic acid molecule. In someembodiments, the two or more immobilized capture probes compriseantibodies that bind the target analyte. In some embodiments, the targetanalyte is a protein.

In some embodiments, the one or more surfaces include a plurality ofsurfaces. In some embodiments, each of the plurality of surfacescomprises a subset of the two or more immobilized capture probes.

In some embodiments, the voltage is a direct current voltage. In someembodiments, the voltage is an alternating current voltage.

In another aspect, a system for detecting a presence of a target analytecomprises at least one sensor comprising one or more surfaces betweentwo or more electrodes, wherein the one or more surfaces comprise two ormore immobilized capture probes; a solution chamber in fluidcommunication with the one or more surfaces, wherein the solutionchamber is configured to retain a solution containing or suspected ofcontaining the target analyte and a non-target analyte, under conditionssufficient to permit the target analyte and non-target analyte to eachbind to a given one of the two or more immobilized capture probes; and acontroller that is operably coupled to the two or more electrodes,wherein the controller is programmed to (i) apply a voltage across thetwo or more electrodes that is sufficient to release the non-targetanalyte, but not the target analyte, from a given one of the two or moreimmobilized capture probes, and (ii) detect a signal indicative of apresence and/or binding energy of the target analyte on the one or moresurfaces. In some embodiments, the target analyte and/or the non-targetanalyte is a charged analyte. In some embodiments, the voltage acrossthe two or more electrodes is sufficient to exert an applied force of atleast 1 piconewton on the non-target analyte.

In some embodiments, the at least one sensor comprises an array ofsensors. In some embodiments, the array of sensors comprises at leastabout 10 sensors. In some embodiments, each sensor of the array ofsensors is independently addressable.

In another aspect, an apparatus for detecting a presence of an analytecomprises a fluidic chamber adapted to contain an electrolyte; two ormore electrodes capable of generating an electric field within thefluidic chamber; at least one surface comprising an immobilized captureprobe capable of binding the analyte, wherein the at least one surfaceis between the two or more electrodes and in contact with the fluidicchamber, and wherein the at least one surface comprises an electricallyconducting layer and an electrically insulating layer; and at least onefield confining feature proximal to the at least one surface, which atleast one field confining feature is capable of concentrating anelectric field surrounding the at least one field confining feature to astrength of at least about 10³ volts per meter (V/m). In someembodiments, the analyte is a charged analyte. In some embodiments, theat least one field confining feature is capable of concentrating theelectric field surrounding the at least one field confining feature to astrength of less than about 10⁵ V/m. In some embodiments, the apparatusfurther comprises a controller operably coupled to the two or moreelectrodes, wherein the controller is programmed to apply a voltageacross the two or more electrodes that is sufficient to generate anapplied force on the analyte. In some embodiments, the voltage appliedis sufficient to generate an applied force of at least about 1piconewton (pN) on the analyte. In some embodiments, the voltage issufficient to generate an applied force of at least about 20 pN on theanalyte.

In some embodiments, the immobilized capture probe is capable of bindingto the analyte to form a probe-analyte complex on the at least onesurface. In some embodiments, the immobilized capture probe is capableof binding to the analyte via non-covalent interaction(s). In someembodiments, the non-covalent interaction(s) are disruptable through anapplied force generated upon the application of a voltage across the twoor more electrodes. In some embodiments, the immobilized capture probeis proximal to the at least one field confining feature. In someembodiments, the at least one field confining feature is a plurality offield confining features.

In some embodiments, the immobilized capture probe is an antibody. Insome embodiments, the analyte is an antigen. In some embodiments, theimmobilized capture probe is a nucleic acid molecule. In someembodiments, the analyte is a nucleic acid molecule.

In some embodiments, the analyte has a charge of less than about 10⁴ e⁻.In some embodiments, the analyte has a charge of greater than about 10e⁻. In some embodiments, the apparatus further comprises a controlleroperably coupled to the two or more electrodes, wherein the controlleris programmed to apply a potential difference of less than about 100volts (V) between the two or more electrodes.

In some embodiments, the at least one surface provides a wall of thefluidic chamber and the field confining features is an orifice in thesurface. In some embodiments, the field confining feature comprises anorifice in the at least one surface. In some embodiments, the fluidicchamber comprises a top portion and a bottom portion partitioned by theat least one surface, and wherein the field confining feature is anorifice in the at least one surface. In some embodiments, the orificeextends through the surface. In some embodiments, the orifice is anindentation, a well, a pore, a channel, a gap, or a slit. In someembodiments, the at least one field confining feature has a diameter ofless than about 50 micrometers. In some embodiments, the at least onefield confining feature has a diameter of less than about 50 nanometers.In some embodiments, the field confining feature has an aspect ratio ofat least about 0.1. In some embodiments, the aspect ratio is the ratioof the longest dimension of the field confining feature to the shortestdimension of the field confining feature. In some embodiments, theaspect ratio is the ratio of the width of the field confining feature tothe depth of the field confining feature. In some embodiments, the fieldconfining feature has a sharp edge. In some embodiments, the fieldconfining feature is an elevated portion of the at least one surface.

In some embodiments, the electrically conducting layer is incommunication with the electrolyte. In some embodiments, theelectrically conducting layer is electrically isolated from theelectrolyte, and wherein, during use, the electrically conducting layeris electrically biased. In some embodiments, the electrically conductinglayer comprises platinum. In some embodiments, the apparatus furthercomprises a sensing electrode configured to measure a charge signalassociated with the presence of the analyte.

In some embodiments, the sensing electrode is proximal to the at leastone field confining feature. In some embodiments, the apparatus furthercomprises a light source and a detector configured to detect an opticalsignal associated with the presence of the analyte. In some embodiments,the optical signal is a fluorescence signal. In some embodiments, thefluorescence signal is provided by a fluorescent probe covalently ornon-covalently attached to the analyte. In some embodiments, theapparatus further comprises a detector configured to detect a surfaceplasmon resonance signal associated with the presence of the analyte.

In some embodiments, the at least one surface is substantially planar.

In some embodiments, the at least one surface is part of an array ofsensors, wherein each sensor of the array comprises a field confiningfeature and a plurality of immobilized capture probes. In someembodiments, the plurality of immobilized capture probes of a givensensor of the array are proximal to the field confining feature for thegiven sensor of the array. In some embodiments, the plurality ofimmobilized capture probes of a given sensor of the array is clonal. Insome embodiments, a given sensor of the array comprises a plurality ofimmobilized capture probes that is unique relative to another sensor ofthe array. In some embodiments, a distance between a given sensor of thearray and a nearest neighbor sensor is at least about 50 nanometers(nm). In some embodiments, the distance between the given sensor of thearray and the nearest neighbor sensor is at least about 150 nm. In someembodiments, a distance between a given sensor of the array and anearest neighbor sensor is less than about 50 micrometers. In someembodiments, the array comprises at least about 10 sensors.

In another aspect, a method for analyzing a binding energy between atarget analyte and a capture probe comprises (a) activating at least onesensor comprising one or more surfaces between two or more electrodes,wherein the one or more surfaces comprises an immobilized capture probe;(b) bringing the one or more surfaces in contact with a solutioncontaining or suspected of containing a target analyte under conditionssufficient to permit the target analyte to bind to the immobilizedcapture probe; (c) applying a voltage across the two or more electrodesthat is sufficient to exert an applied force of at least about 1piconewton (pN) on the target analyte to release the target analyte formthe immobilized capture probe; and (d) detecting a signal indicative ofthe binding energy between the target analyte and the capture probe. Insome embodiments, the applied force is at least about 10 pN. In someembodiments, the voltage is sufficient to release the target analyte,but not a non-target analyte, from the one or more surfaces. In someembodiments, the voltage is sufficient to generate an electric fieldhaving a strength of at least about 10³ volts per meter.

Another aspect of the present disclosure provides a computer-readablemedium comprising machine executable code that, upon execution by one ormore computer processors, implements any of the methods above orelsewhere herein.

Another aspect of the present disclosure provides a computer systemcomprising one or more computer processors and computer memory coupledthereto. The computer memory comprises machine executable code that,upon execution by the one or more computer processors, implements any ofthe methods above or elsewhere herein.

Additional aspects and advantages of the present disclosure will becomereadily apparent to those skilled in this art from the followingdetailed description, wherein only illustrative embodiments of thepresent disclosure are shown and described. As will be realized, thepresent disclosure is capable of other and different embodiments, andits several details are capable of modifications in various obviousrespects, all without departing from the disclosure. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings or figures (also “FIG.” and “FIGS.” herein), ofwhich:

FIG. 1A shows an example of a cross-sectional profile view of anapparatus of the present disclosure; FIG. 1B shows an example of across-sectional profile view of an apparatus of the present disclosure;

FIG. 2A shows an example of the effect of sensor array size on electricfield strength; FIG. 2B shows an example of the effect of aspect ratioon the inter-sensor variation in electric field strength;

FIG. 3 shows an example of the electric field produced without anelectrically conducting layer;

FIG. 4 shows an example of the electric field produced with anelectrically conducting layer (e.g., field terminating plane);

FIG. 5 shows an example of an apparatus of the present disclosureadapted for optical detection of charged analytes;

FIG. 6A shows an example of the magnitude of the fluorescent signal as afunction of applied voltage for operation of the apparatus of FIG. 5;FIG. 6B shows an example of the operation of the apparatus of FIG. 5 ata first voltage; FIG. 6C shows an example of the operation of theapparatus of FIG. 5 at a second voltage; FIG. 6D shows an example of theoperation of the apparatus of FIG. 5 at a third voltage; FIG. 6E showsan example of the operation of the apparatus of FIG. 5 at a fourthvoltage;

FIG. 7 shows an example of an apparatus of the present disclosureadapted for electrical detection of charged analytes;

FIG. 8A shows an example of the magnitude of sensor voltage as afunction of applied voltage for operation of the apparatus of FIG. 7;FIG. 8B shows an example of the operation of the apparatus of FIG. 7 ata first voltage; FIG. 8C shows an example of the operation of theapparatus of FIG. 7 at a second voltage; FIG. 8D shows an example of theoperation of the apparatus of FIG. 7 at a third voltage; FIG. 8E showsan example of the operation of the apparatus of FIG. 7 at a fourthvoltage;

FIG. 9 shows an example of an apparatus of the present disclosureadapted for plasmonic detection of charged analytes;

FIG. 10 shows an example of an array of the present disclosure forsensing the presence and binding force of nucleic acid molecules;

FIG. 11A shows an example of the method of the present disclosure beingused to measure the binding force of a complimentary nucleic acidmolecule; FIG. 11B continues from FIG. 11A and shows an example of themethod of the present disclosure being used to measure the binding forceof a complimentary nucleic acid molecule;

FIG. 12A shows an example of the method of the present disclosure beingused to measure the binding force of a nucleic acid molecule having asingle base pair mismatch to the capture probe; FIG. 12B continues fromFIG. 12A and shows an example of the method of the present disclosurebeing used to measure the binding force of a nucleic acid moleculehaving a single base pair mismatch to the capture probe;

FIG. 13A shows an example of the method of the present disclosure beingused to measure the binding force of a mixture of nucleic acid moleculeshaving various amounts of complimentarity to the capture probe; FIG. 13Bcontinues from FIG. 13A and shows an example of the method of thepresent disclosure being used to measure the binding force of a mixtureof nucleic acid molecules having various amounts of complimentarity tothe capture probe;

FIG. 14 shows an example of a multiplexed immunoassay where secondaryantibodies have an attached nucleic acid to increase and control theamount of charge associated with the analyte;

FIG. 15 shows an example of performing the method of the presentdisclosure using the apparatus shown in FIG. 14;

FIG. 16 shows an example of the attachment of a nucleic acid molecule toa surface of the present disclosure;

FIG. 17 shows an example of an apparatus of the present disclosure; and

FIG. 18 shows an example of a computer system for operation of theapparatus of the present disclosure.

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and describedherein, it will be obvious to those skilled in the art that suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions may occur to those skilled in the art withoutdeparting from the invention. It should be understood that variousalternatives to the embodiments of the invention described herein may beemployed.

An externally applied electric field in an aqueous solution can apply aforce on a charged particle (e.g., molecule or analyte) in the solution.Such force on the charged particle (F) can be proportional to theapplied electric field (E) and the net electric charge of the particle(q) according to the equation F=qE.

Using the linear relation among these parameters, the present methodcorrelates the applied force to the input parameters (e.g., appliedexternal biases). Provided herein is an apparatus that may utilize suchforce to perform a force spectroscopy and force differentiation assay.The assay can be performed on antigen-antibody bonds or used for nucleicacid analysis (e.g., single nucleotide polymorphism (SNP) analysis). Insome cases, the binding force between the analyte and a capture probecan be from about 1 piconewton (pN) to about 400 pN.

Given an example of a charged particle having a charge of 100e− (e.g.,equivalent to a nucleic acid with 100 bases), the external electricfield strength used to disrupt some bonds of interest are shown asfollows in Table 1.

TABLE 1 Electric field needed to disrupt bonds ofinterest (assuming 100e− of charge on the labeling molecule). EfieldForce needed Molecule [pN] [V/m] Streptavidin Biotin 141 8.8E+0614mer SNP (CCAAACCAACCACC) 178 1.1E+0714mer SNP 1 base change 8^(th) C to G 130 8.1E+0614mer SNP 1 base change 8^(th) C to A 133 8.3E+06 Biotin-Antibiotin 1127.0E+06 Ferritin-Antiferritin 49 3.1E+06

The field strengths needed may be relatively large, which may bedifficult to generate in an open structure. Therefore, the presentapparatus employs (physical) confinement using (micro or nano-scale)structures to significantly boost the applied electric field strength.These structures are referred to herein as “field confinement features”and can include pores, channels, gaps and slits (e.g., orifices), aswell as raised structures associated with a sensing surface. Nanoporesare a type of field confinement feature which can physically confinetranslocation of molecules, enabling single molecule detection viablocking of the nanopore.

In some cases, an advantage of such physical confinement is theelectrical confinement that comes with it. When designed with theorifice being the smallest feature, the orifice can dominate theelectrical characteristics of the entire system. The potential drop(thus the electric field) can occur primarily near the orifice and less(to very little) elsewhere. When the orifice is small (e.g., micro ornano scale) such confinement can generate a large electric field withrelatively low applied bias. For example, a synthetic nanopore on a 100nm thick SiN_(x) membrane can generate about 10,000,000 V/m of electricfield across it with application of 1 V. The present disclosureencompasses any size, shape or arrangement of field confining features(e.g., orifices), including the embodiments shown in FIG. 1A and FIG.1B.

By preparing a group of recognition molecules (e.g., capture probes) inadvance, the present method can look for the presence of theirrespective target molecules (e.g., analytes) in a sample by observingthe quantity and force by which the analytes bind to the capture probes.The present method of analyzing such molecular recognition (e.g.,binding) events can be referred to as a binding assay. Labeled (orlabeling) agents can be used for detection of such molecular recognitionevents. Non-specific binding may present a challenge in existing bindingassays. Other limitations of existing binding assays include aninability to multiplex the assay, inability to perform the assay on amicro or nano-scale (e.g., be densely integrated), and inability togenerate sufficient forces.

Examples of existing binding assays include, but are not limited to:enzyme-linked immunosorbent assay (ELISA); sandwich ELISA; proximityligation assay (in situ PLA); force differentiation assays (as describedin Juncker, D., S. Bergeron, et al. (2014) “Cross-reactivity in antibodymicroarrays and multiplexed sandwich assays: shedding light on the darkside of multiplexing.” Current Opinion in Chemical Biology 18: 29-37);micropipettes used in conjunction with optical microscopy (as describedin Evans, E. and K. Ritchie (1997) “Dynamic strength of molecularadhesion bonds.” Biophysical Journal 72(4): 1541-1555); use of laminarfluid flow to generate physical force onto beads of certain sizes (asdescribed in U.S. Pat. No. 7,736,889); ultrasound excitation used toagitate bound molecules (as described in U.S. Pat. No. 6,086,821); useof magnetic field to deliver the forces to intermolecular bonds (asdescribed in U.S. Pat. Nos. 5,445,970; 5,445,971 and 6,180,418); use ofdielectrophoresis (as described in U.S. Patent Application No.2014/0102901); use of atomic force microscopy (AFM) (as described inU.S. Pat. Nos. 5,363,697; 5,992,226; 5,992,226; Florin, E. L., V. T.Moy, et al. (1994) “Adhesion Forces between Individual Ligand-ReceptorPairs.” Science 264(5157): 415-417; Lee, G. U., D. A. Kidwell, et al.(1994) “Sensing Discrete Streptavidin Biotin Interactions withAtomic-Force Microscopy.” Langmuir 10(2): 354-357; Dammer, U., M.Hegner, et al. (1996) “Specific antigen/antibody interactions measuredby force microscopy.” Biophysical Journal 70(5): 2437-2441; Chilkoti,A., T. Boland, et al. (1995) “The relationship between ligand-bindingthermodynamics and protein-ligand interaction forces measured by atomicforce microscopy.” Biophysical Journal 69(5): 2125-2130 and Allen, S.,X. Y. Chen, et al. (1997). “Detection of antigen-antibody binding eventswith the atomic force microscope.” Biochemistry 36(24): 7457-7463); useof synthetic pores (as described in U.S. Pat. No. 8,810,787 andTabard-Cossa, V., M. Wiggin, et al. (2009) “Single-Molecule BondsCharacterized by Solid-State Nanopore Force Spectroscopy.” ACS Nano3(10): 3009-3014); and use of biological pores (as described in Dudko,O. K., J. Mathe, et al. (2007) “Extracting kinetics from single-moleculeforce spectroscopy: Nanopore unzipping of DNA hairpins.” BiophysicalJournal 92(12): 4188-4195 and Tropini, C. and A. Marziali (2007).“Multi-nanopore force Spectroscopy for DNA analysis.” BiophysicalJournal 92(5): 1632-1637), each of which (patent and/or research paper)publications are incorporated herein by reference in their entirety forall purposes.

Various types of cross-reactivity may occur in binding assays, FIG. 14shows some examples. Given a variety of labels or detection probes, anincorrect label or detection probe can bind to the target in addition toa correct label or detection probe, 1410. An incorrect label ordetection probe can bind to the capture probe, 1413. An incorrect labelor detection probe can bind to the correct label or detection probebound to the correct target, 1411. An incorrect label or detectionprobe-target compound can bind to the correct probe, 1414. An incorrecttarget can bind to the correct capture probe which has the correcttarget and correct label or detection probe bound, 1412. An incorrectlabel or detection probe can be gravitationally or coulombicallyadsorbed to the detecting surface in absence of any probes. Each ofthese events can cause an excess of labels or probes to remain on anassay surface resulting in false positives results. Such false positiveresults can be reduced by the apparatus and methods of the presentdisclosure, e.g., by applying a force to a surface that is sufficient toremove undesirable labels or probes or targets and/or by having an apriori expectation of the binding force of the captureprobe-target-label/detection probe or probe-target complex andcorrespondingly measuring the force required to break the complex incomparison with the expected binding force.

In an aspect, the present disclosure describes a method for measuringintermolecular interactions, including but not limited toreceptor/ligand, protein/protein, nucleic acid/protein and nucleicacid/nucleic acid interactions. A method for measuring intermolecularinteractions can involve the simultaneous use of (1) a sensor fordetecting the attachment of target molecules in solution to theirspecific, complementary or near complementary capture molecule bound toa surface (e.g., the hybridization of a nucleic acid in solution to acomplementary or near complementary nucleic acid capture probe on thesurface) and (2) the use an electric field to apply an electrokineticforce on the bound molecule complex (e.g., a target molecule attached orbound to a capture molecule). When the electrokinetic force exceeds theattachment force of the bound molecule complex, the target molecules canbecome detached from the capture molecule. This can occur because theattachment force of the capture molecule to the surface can far exceedthe attachment force of the target molecule to the capture probe. Theelectrokinetic force applied by the electric field is such thatmolecules can be pulled away from the sensor. An electric field may begenerated by applying a voltage on two opposing electrodes on thesurface of the sensor and device or in solution or a combination. Thistarget molecule may have sufficient charge to create a high enoughelectrokinetic force. In some cases, the target molecule can be modifiedwith additional charge to create a higher electrokinetic force on thetarget molecule. As an example, target nucleic acid molecules can bemodified during polymerase chain reaction (PCR) using primers comprisinga polyanionic polymer. In the case of immunoassays, a secondary antibodyor detection antibody can be labeled with nucleic acid or a polyanionicpolymer to enable the application of an electrokinetic force on theantibody in the case that the target molecule has insufficient charge.

The present disclosure also provides several apparatuses capable ofemploying this method, including an all-electronic platform. In anaspect, the present disclosure provides an apparatus for detecting acharged analyte. The apparatus can comprise a fluidic chamber adapted tocontain an electrolyte. The apparatus can further include two or moreelectrodes, such as a first electrode and a second electrode, capable ofproviding an electric field within the electrolyte and a surface locatedbetween the first electrode and the second electrode and in contact withthe fluidic chamber. The surface can include an electrically conductinglayer and an electrically insulating layer. There can be a fieldconfining feature located in proximity to the surface. The fieldconfining feature can concentrate the electric field to a strength of atleast about 10³ volts per meter (V/m), at least about 10⁴V/m, at leastabout 10⁵V/m, at least about 10⁶ V/m, at least about 10⁷V/m, at leastabout 10⁸ V/m, at least about 10⁹V/m or greater in proximity to thefield confining feature.

In another aspect, the present disclosure provides a method fordetecting binding of a charged analyte. The method can include providinga device having two or more electrodes, such as a pair of electrodes,and a surface between the pair of electrodes. The surface can have acapture probe tethered to a surface at a sensing location. The methodcan further include contacting the surface with a mixture of chargedanalytes, where the mixture comprises a target charged analyte and anon-target charged analyte. The charged analytes can form non-covalentbonds with the capture probe. The method can further comprise applying afirst voltage across the pair of electrodes, which first voltage resultsin a first electric field that exerts a first applied force of at least1 piconewton (pN) on the charged analytes that are bound to the captureprobe, thereby breaking non-covalent bonds between the capture probe andthe non-target charged analyte. The method can then include detectingthe presence of the target charged analyte at the sensing location. Insome cases, the first electric field has a strength of at least about10³ volts per meter (V/m), at least about 10⁴V/m, at least about 10⁵V/m,at least about 10⁶ V/m, at least about 10⁷V/m, at least about 10⁸ V/m,at least about 10⁹V/m or greater in proximity to the sensing location.

In some cases, the method can further comprise applying a second voltageacross the pair of electrodes, which second voltage results in a secondelectric field that exerts a second applied force of greater than 1 pNon the charged analytes that are bound to the capture probe, therebybreaking non-covalent bonds between the capture probe and the targetcharged analyte. The method can include detecting the absence of thetarget charged analyte at the sensing location.

Turning now to the figures, FIG. 1A shows an example of across-sectional profile view of an apparatus of the present disclosure.The apparatus can include a fluidic chamber adapted to contain anelectrolyte 101. A first electrode 102 can be located in the electrolyteand form a circuit with a second electrode 103, across which electrodesa voltage can be applied by a voltage source 104. In this case, thesecond electrode forms one of the walls of the fluidic chamber. In somecases (e.g., see FIG. 1B), the fluidic chamber is bisected by a membraneand the first and second electrodes are on opposing sides of themembrane. A surface is laid in proximity to or upon the second electrodein this embodiment. The surface can include an electrically insulatinglayer 105 and an electrically conducting layer 106. In some cases, theelectrically conducting layer is exposed to the electrolyte 101. Thesurface can have field confinement features 107 in proximity to thesurface (e.g., within or upon the surface). The field confinementfeatures can increase the magnitude of an applied electric field inproximity to the field confinement features.

The apparatus can use an electric field to draw an anchored captureprobe and its attached target into a pore to detect the total chargeusing an auxiliary sensor. The difference in charge between the capturemolecules in their non-hybridized (e.g., not bound to target) andhybridized states (e.g., bound to target) can be used to determine thepresence of bound molecules. This architecture can be well suited forprobing the binding energy of a molecule pair. While measuring thepresence of the molecule pair under the influence of an electric field,the electric field can be gradually increased until the electrokineticforce exceeds the binding energy of the molecule pair. Once the targetmolecule detaches, a change in charge in the sensing area can bedetected and that change can be used to determine the binding voltage ofthe pair.

FIG. 1B shows an example of a cross-sectional profile view of anapparatus of the present disclosure where the sensor surface bisects thefluidic chamber. The fluidic chamber of FIG. 1A and FIG. 1B can be amicrofluidic chamber (e.g., having a smallest dimension of less thanabout 1 millimeter). As with FIG. 1A, the apparatus can include anelectrolyte 101, a first electrode 102, a second electrode 103, avoltage source 104, an electrically insulating layer 105, anelectrically conducting layer 106 and a field confinement feature 107.

Various factors may affect the degree of confinement of a confinementfeature (e.g., orifice). The design of the confinement features, such asaspect ratio and density of features, may affect the degree ofconfinement. Higher aspect ratios may help to reduce the effects of afringing field. For dense packing of the confining features, the overlapin fringing field can reduce the effect of field confinement. In somecases, the overlapping of fringing field between features can be reducedby providing a region for the fields to terminate before overlapping,such as a conducting surface near the confinement feature. Such asurface can effectively pin the potential near the orifice to allow forfurther enhancement of the electric field and thus the generatedelectrokinetic force. An in silico study of the effect of such a fieldterminating location along with an investigation into the effects offeature aspect ratio is shown herein. The presence of a conductivesurface layer may both enhance single feature's ability to confine theelectric field and be very effective against countering the reductionfrom integration of confinement features into an array.

As described herein, the size, shape, arrangement and number of fieldconfinement features can have an effect on the magnitude of the electricfield created in proximity to the field confinement features and theamount of force that can be applied to the charged analyte. FIG. 2Ashows an example of the effect of sensor array size on electric fieldstrength. As seen here, the electrically conductive layer (also referredto as the potential pinning or field terminating layer/plane) may helpto create a stronger electric field, particularly as the number ofsensing locations (e.g., wells) increases. The electric field in thiscase may be relatively constant at about 1.5×10⁶ V/m when anelectrically conducting layer is used 201, while the electric field inthe absence of the conducting layer 202 may be both lower in magnitudeand decreases as the well array size increases. The horizontal axis isnumber of sensing locations on a side of a square array (e.g., a singlelocation, a 3×3 array, a 5×5 array and a 7×7 array). The electric fieldsdepicted in FIG. 2A, FIG. 2B, FIG. 3 and FIG. 4 are achieved withconfinement features having a diameter of 1 μm and spaced 4 μm apart.

FIG. 2B shows an example of the effect of aspect ratio on theinter-sensor variation in electric field strength. The systemarchitecture is an array of wells as shown in FIG. 1A, where the aspectratio is defined as the width of the well (e.g., along the surface)divided by the depth of the well (e.g., into the surface). As shown, theelectric field strength variation (as measured in %) 203 increases withincreasing aspect ratio. The electric fields at the bottoms of themicrowells are compared to determine the variation. In this example, thevariation in electric field approaches 0.0% (e.g., all sensing locationshave the same field) at an aspect ratio of 1:1. In some embodiments, theaspect ratio is less than about 5:1, less than about 4:1, less thanabout 3:1, less than about 2:1, less than about 1:1, or less than about1:2.

The effect of the field terminating layer is shown graphically in FIG. 3and FIG. 4. Referring to FIG. 3, the system includes an insulating layer301, an electrolyte 302, and a first electrode 303. A voltage can beapplied between the first electrode and second electrode (not shown) tocreate an electric field that is concentrated in the field confinementfeatures (wells in this case). FIG. 3 shows a series of three profileviews of sensor arrays. The magnitude of the electric field decreases asthe number of wells increases from a single well 304, to a 3×3 array of9 wells 305, to a 7×7 array of 49 wells 306.

FIG. 4 shows a system similar to FIG. 3 including an insulating layer301, an electrolyte 302, and a first electrode 303. The system alsoincludes an electrically conducting layer 400 disposed upon theinsulating layer. In comparison to FIG. 3 without the electricallyconducting layer, the magnitude of the electric field is both strongerand does not diminish with increasing array size. The magnitude of theelectric field is shown with shading (darker being stronger) for asingle sensor 401, a 3×3 array 402 and a 7×7 array 403.

In some embodiments, the applied electrokinetic force on the chargedanalyte ranges from 0 pN to 5000 pN. In some instances, the electricfield confining feature (e.g., an orifice) and/or conducting potentialpinning plane can be used to enhance the electrokinetic force. In somecases, the applied force is sufficient to dissociate the analytes fromthe capture probes (e.g., receptor/ligand, protein/protein, nucleicacid/protein or nucleic acid/nucleic acid interactions) sequentially(e.g., in the order of their binding strengths). The method can be ableto judge whether molecules are correctly (specifically) or incorrectly(non-specifically) bound to their intended targets.

In some embodiments, the apparatus and methods of the present disclosureare used to detect and probe the binding force of a single nucleotidepolymorphism (SNP) or to analyze immunoassays (e.g., sandwichimmunoassays). In some cases, more than one sensor can be integrated ina single chip to allow for parallel analysis of SNPs, immunoassay andsandwich immunoassays by themselves or simultaneously.

The methods of the present disclosure can involve detecting chargedanalytes by any suitable approach, such as, for example, opticaldetection, electrical or electrostatic detection, or plasmonicsdetection.

FIG. 5 shows an example of an apparatus of the present disclosureadapted for optical detection of charged analytes (e.g., using amodified fluorescence based DNA microarray). The apparatus can include atop substrate 501 and a bottom substrate 515 (e.g., which can be anoptically transparent material), as well as fluidic walls 518 thatdefine a fluidic chamber. In some cases, the chamber is microfluidic(e.g., having micrometer dimensions) and contains an electrolyte 502. Avoltage source 523 can be connected by electrical connectors 520 to afirst electrode 522 and a second electrode 506. The electrode can beoverlaid by a surface having an insulating layer 516 and a conductinglayer 517. The surface can be interrupted by a plurality of fieldconfinement features, which in this embodiment are clustered into afirst sensing location 507 and a second sensing location 514, whichsensing locations are pixels in the optical detection system shown here.The optical detection system can include a light source 512, anexcitation filter 511, a dichroic mirror 509 and an emission filter 508.The light can pass through these components as well as an optics module513 (e.g., containing lenses) that directs light to the sensinglocations at an excitation wavelength and receives light from thefluorescent label 503 at an emission wavelength. The emission radiationcan be imaged using an imaging system 510 (e.g., containing a camera).Capture probes (e.g., antibodies) can be tethered in proximity to thesensing locations. In this case, a first antibody 505 is tethered to thesurface (e.g., to the electrode) at the first sensing location 507 and asecond antibody 519 is tethered at the second sensing location 514. Afirst target antigen 504 binds to the first antibody 505 (e.g., captureprobe) and a secondary antibody 521 having a fluorescent probe 503 canbind to the target antigen in order to concentrate a fluorescent signalat the sensing locations.

The bottom surface can act like the surface of a traditional nucleicacid microarray and is packaged with a fluidic channel on top. Both thetop surface of the microarray and the bottom surface of the fluidicchannel can be modified to have an electrode. Either one or bothelectrode surfaces can be a transparent conducting material, for exampleindium tin oxide (ITO) to allow for simultaneous imaging of the surfaceand the application of an electric field. The process of detection isshown in FIG. 6A to FIG. 6E. For clarity, it may be assumed that onlyevents on the surface lead to a fluorescent signal and there is verylittle background. In practice, this can be accomplished by firsthybridizing the target nucleic acid on the microarray slide and washingprior to testing. As the voltage is increased, the electrokinetic forceon the hybridized nucleic acid may increase until nucleic acid is pulledoff. This pull-off voltage can be related to the binding energy of thetarget analyte to the capture probe, which is identified as the bindingenergy voltage (VBE). This can be used to discriminate between a targetthat may be a correct complementary match and a target that has a singleor plurality of mutations and is not completely complementary to thecapture probe. A target that is not completely complimentary to thecapture probe may still hybridize to the probe. The methods of thepresent disclosure can be used to identify this hybridization between atarget that is not completely complementary to the capture probe (e.g.,due to the target detaching at a lower voltage). This method can beemployed to identify mixed mutation samples on a single capture site.

FIG. 6A shows an example of the magnitude of the fluorescent signal as afunction of applied voltage for operation of the apparatus of FIG. 5.The applied voltage can be increased over time and the fluorescenceoutput at the first pixel 601 and the second pixel 602 can be monitored.The signal begins relatively higher at lower applied voltages as analyteis bound both specifically and non-specifically and decreases accordingto the curves shown in FIG. 6A as the voltage is increased, providing aforce that pulls the charged analytes from the capture probes. A seriesof drawings are provided which clarify the binding of the analytes atvarious applied voltages; with the lowest voltage at position 603corresponding to FIG. 6B, the third highest voltage at position 604corresponding to FIG. 6C, the second highest voltage at position 605corresponding to FIG. 6D, and the highest voltage at position 606corresponding to FIG. 6E.

FIG. 6B shows an example of the operation of the apparatus of FIG. 5 ata first voltage where initial binding occurs. The first pixel 611 has aplurality of the first capture antibodies 609 tethered to it and thesecond pixel 612 has a plurality of the second capture antibodies 610tethered to it. The charged analyte 608 can bind specifically (e.g., athigh strength) to the first capture antibody and can bindnon-specifically (e.g., at low strength) to the second capture antibody.As shown in FIG. 6A, a fluorescent signal can be detected at both thefirst and second pixel, however, since the magnitudes of the signals aresimilar at the first voltage, specific versus non-specific bindingcannot be easily differentiated at the first applied voltage. Therefore,the applied voltage can be increased to a second voltage.

FIG. 6C shows an example of the operation of the apparatus of FIG. 5 ata second voltage where non-specific components detach 613. Thedetachment from the second capture antibody can be due to the appliedforce exerted on the charged analyte by the electric field in proximityto the confinement features. In this case, some non-specifically boundanalytes 614 remain bound at the second voltage, which can be detachedby increasing the applied voltage further.

FIG. 6D shows an example of the operation of the apparatus of FIG. 5 ata third voltage. The remaining non-specific binders detach 614. In somecases, some true positives 615 (e.g., specifically bound analytes)detach as well. As shown in FIG. 6E, the applied voltage can beincreased yet further to a fourth voltage where all components detach.The third and/or fourth voltages can be used to determine the bindingforce between the target antigen and the capture probe.

In some cases, the charged analytes can be electronically detected. FIG.7 shows an example of an apparatus of the present disclosure adapted forelectrical detection of charged analytes. The apparatus can include afluidic chamber 702 (e.g., microfluidic) that is bisected by a surface.The surface can include several layers including a substrate 707, aninsulating layer 704 (e.g., a dielectric membrane) and a fieldterminating layer 712 (e.g., a conducting metal such as platinum,copper, aluminum or silver). An applied voltage can be applied using avoltage source 708 in electrical communication with a first electrode701 and a second electrode 706. A sensing electrode can also be locatedin proximity to the surface. In this case, a first sensing electrode 705surrounds the first field confinement feature 713 and a second sensingelectrode 709 surrounds the second field confinement feature 714. Thesensing electrodes can be individually addressable. The binding ofcharged analytes in proximity to the first field confinement feature canbe monitored by a first voltage output 703. The binding of chargedanalytes in proximity to the second field confinement feature can bemonitored by a second voltage output 710.

FIG. 8A shows an example of the magnitude of the electrical signal as afunction of applied voltage for operation of the apparatus of FIG. 7.The applied voltage can be increased over time and the voltage outputnear the first confinement feature 801 and near the second confinementfeature 802 can be monitored. The signal begins relatively lower atlower applied voltages as analyte is bound both specifically andnon-specifically and pulled into the orifice (e.g., field confinementfeature) and eventually detach. A series of drawings are provided whichclarify the binding of the analytes at various applied voltages; withthe lowest voltage at position 803 corresponding to FIG. 8B, the thirdhighest voltage at position 804 corresponding to FIG. 8C, the secondhighest voltage at position 805 corresponding to FIG. 8D, and thehighest voltage at position 806 corresponding to FIG. 8E.

FIG. 8B shows an example of the operation of the apparatus of FIG. 7.The apparatus has a first orifice 807 and a second orifice 808. A firstcapture probe 809 is tethered in proximity to the first orifice 807 anda second capture probe 810 is tethered in proximity to the secondorifice 808. In this case, the analyte 812 itself is not charged. It canbecome charged by associating with a charge label 811. The charge labelcan be covalently or non-covalently attached to the analyte, and theanalyte may thereby become a charged analyte. The charge label can beafixed to a secondary antibody that also binds to the analyte. Theanalyte can bind to the capture probes and the secondary antibody at alow (or zero) applied voltage 803.

As shown in FIG. 8C, the applied voltage can be increased to a secondvoltage sufficient to pull the charged analytes into the orifice 813. Anelectrical signal can be detected at both the first and second orifice,however, since the magnitudes of the signals are similar at the secondvoltage, specific versus non-specific binding cannot be easilydifferentiated at the second applied voltage. Therefore, the appliedvoltage can be increased to a third voltage.

FIG. 8D shows an example of the operation of the apparatus of FIG. 7 ata third voltage where non-specific components detach 814. The detachmentfrom the second capture antibody can be due to the applied force exertedon the charged analyte by the electric field in proximity to theconfinement feature. As shown in FIG. 8A, there is a difference insensor voltage signal between the first orifice and the second orifice,therefore the third voltage can distinguish between specific andnon-specific binding of the analyte in proximity to the field confiningfeature.

FIG. 8E shows an example of the operation of the apparatus of FIG. 7 ata fourth voltage. The true positives (e.g., specifically bound analytes)can detach 815. The third and/or fourth voltages can be used todetermine the binding force between the target antigen and the captureprobe.

In some embodiments, the detection of target molecules is based onplasmonics. An example using a surface plasmon resonance imaging setup(SPRI) is provided herein. The gold film of an SPRI chip can be used forone electrode and a conducting film on the bottom of the fluidic cap canbe used as a second. In some cases, an electrode for this system may notbe transparent.

The methods disclosed herein can be used for probing cross-reactive andnon-specific binding events in immunoassays. Non-specific andcross-reactive antibodies may have much lower binding energies thanspecific antibodies. By applying an electrokinetic force, theapproximate binding energy voltage of the molecular pairs can bedetermined and non-specific and cross-reactive groups can be identifiedand removed in order to identify the presence of specific antibodies. Insome embodiments, the capture probes are antibodies tethered to apolymer chain such as DNA.

In some embodiments, the target analytes are directly detected by thesensor. In such case, cross-reactivity or non-specific binding can occurwhen a secondary antigen is bound to the surface. The appliedelectrokinetic force can then act on the analytes themselves, probingthe binding strength between the analytes and the capture probe. As theelectrokinetic force is increased, the cross-reactive secondary analyte,more weakly bound to the capture probe than the target analyte, candissociate at a smaller applied electrokinetic force than the targetantigen.

In some cases, a sensing or detection antibody is used to enhance thedetectability of the antigens. The sensing antibody can be modified toenhance the detectability by the sensor (e.g., by conjugation to anucleic acid or protein). In embodiments where a sensing antibody isused, possible reactions include, but are not limited to: (a) correctinteraction where the capture antibody is attached to the target antigenand the correct sensing antibody, modified or otherwise, binds to thetarget antigen; (b) a cross-reaction where a secondary antibody binds tothe target antigen; (c) a cross-reaction where a secondary antibodybinds to the capture antibody; (d) a cross-reaction where a secondaryantigen binds to the capture antibody and a secondary antibody binds tothe secondary antigen; (e) a secondary antibody binding to the captureantibody in absence of the target antigen; and (f) a secondary antigenbinding to the capture antibody and a secondary antibody binds to thesecondary antigen. The increasing electrokinetic force can remove thecross-reactive components, such as described in (b)˜(f), earlier thanthe correct interaction, such as described in (a).

FIG. 9 shows an example of an apparatus of the present disclosureadapted for plasmonic detection of charged analytes. The apparatusincludes charged analytes 901 to be detected, which in this case arenucleic acid molecules. The plasmonic detection system can include alight source 904, a polarizer 903, a prism 902, a metal (e.g., gold)film 908, an optical filter 906 and a detector 905. The charged analytes901 can be suspended in an electrolyte confined in a fluidic chamber909. The charged analytes 901 can hybridize to the capture probes 907,thereby becoming detectable by the plasmonic system.

In some embodiments of the present disclosure, the detection of targetmolecules is based on ionic current blockage of a single pore or porousstructure. Some instances of the present disclosure include methods andapparatuses where the applied electrokinetic force is enhanced by thepresence of membranes whose thickness ranges from about 0.08 nanometer(nm) to about 1 millimeter (mm). These apparatuses also include one ormore electrodes which are embedded within the membrane that are used tosense the presence or absence of the target molecule. In some cases, theembedded electrodes may be used to control the physical location of thecapture probes and/or the target molecules. These apparatuses can alsoinclude various orifices through which the electrokinetic force isconcentrated. These orifices can be any opening in the membraneincluding pores, slits or gates of various sizes. Via these orifices,the electric field (generating the electrokinetic force) may be directedin any desired direction. In some cases, the surface of the apparatus iscoated with material to change its surface charge in solution, suchmaterials include but are not limited to Silicon Dioxide (SiO₂), SiliconNitride (SiN_(x)), Hafnium Dioxide (HfO₂), Zirconium Dioxide (ZrO₂),Aluminum Oxide (Al₂O₃) and Titanium Dioxide (TiO₂).

FIG. 10 shows an example of an array of the present disclosure forsensing the presence and binding energy of nucleic acid molecules. Thesystem includes a fluidic chamber 1000 containing a buffer 1002 andopposed by a pair of transparent electrodes 1004 capable of creating anelectric field. In this case, the field confinement features are notshown. The charged antigens contain fluorescent labels 1006 and canhybridize to the capture probes 1008. Nucleic acids are typicallynegatively charged, so no secondary charge label may be needed.

FIG. 11A shows an example of the method of the present disclosure beingused to measure the binding force of a complimentary nucleic acidmolecule. The graphic depicts a series of three states of the system(top to bottom) having different relative applied voltages (dashedlines) and fluorescent signals (solid lines). The target analytes areinitially not bound to the capture probes 1100 and a baseline signal isdetected 1102. The hybridization of the analytes 1104 to the captureprobes increases the signal 1106. Application of a linearly increasingapplied voltage 1108 creates an electric field 1110 and an applied forceon the charged analytes, but does not initially affect the signal. FIG.11B continues from FIG. 11A and shows the effect of continuing toincrease the applied voltage. As the electrokinetic force reaches alevel close to the binding force between the analyte and the captureprobe, some of the target analytes can dissociate 1112 and the signalcan begin dropping 1114. As the voltage continues to increase,eventually all of the analytes can dissociate 1116 and the signal canreturn to baseline. The binding energy voltage can be defined as thevoltage where half of the target analytes are released from the surface1118.

FIG. 12A and FIG. 12B are similar to FIG. 11A and FIG. 11B, but in thisinstance show an example of the method of the present disclosure beingused to measure the binding force of a nucleic acid molecule having asingle base pair mismatch to the capture probe (e.g., a singlenucleotide polymorphism (SNP)). The graphic depicts a series of threestates of the system (top to bottom) having different relative appliedvoltages (dashed lines) and fluorescent signals (solid lines). Thetarget analytes are initially not bound to the capture probes 1200 and abaseline signal is detected 1202. The hybridization of the analytes 1204to the capture probes increases the signal 1206. Application of alinearly increasing applied voltage 1208 creates an electric field 1210and an applied force on the charged analytes, but does not initiallyaffect the signal. With reference to FIG. 12B, the mismatched analytedissociates from the capture probe 1212 at a relatively lower voltagecompared with the voltage at which a perfectly complimentary analytedissociates (see 1112 in FIG. 11B). Also, all mismatched analytesdissociate at a relatively lower voltage 1214 and the binding energyvoltage 1216 is relatively lower compared with an analyte not having theSNP. The difference in signal versus applied voltage behavior betweenFIG. 11B and FIG. 12B can be used to identify the presence of thepolymorphism. Note that the embodiment described herein is able todistinguish between two or more nucleic acid molecules at a singlesensing location using a single capture probe.

The methods described herein can also be used to identify a plurality ofdifferent analytes in a mixture. For example, FIG. 13A and FIG. 13B showthe method of the present disclosure being used to measure the presenceof and/or binding force of a mixture of nucleic acid molecules havingvarious amounts of complementarity to the capture probe. The graphicdepicts a series of three states of the system (top to bottom) havingdifferent relative applied voltages (dashed lines) and fluorescentsignals (solid lines). The target analytes are initially not bound tothe capture probes 1300 and a baseline signal is detected 1302. Thehybridization of the analytes 1304 to the capture probes increases thesignal 1306. Application of a linearly increasing applied voltage 1308creates an electric field 1310 and an applied force on the chargedanalytes, but does not initially affect the signal. Turning attention toFIG. 13B, the voltage signal displays two inflection points and twobinding energy voltages, 1316 and 1318, corresponding to, first,dissociation of the mismatched nucleic acid analytes 1312 anddissociation of the completely complimentary nucleic acid analytes fromthe capture probes 1314, respectively.

FIG. 14 shows an example of a multiplexed immunoassay, which depicts theissue of cross reactivity when trying to test for multiple analytes withmultiple antibodies. Some instances of the assay use secondaryantibodies 1400 that have an attached nucleic acid 1402 (e.g., toincrease and control the amount of charge associated with the analyte1404, which can be bound to a capture probe 1406 tethered to a surface1408). FIG. 14 shows six different types of cross-reactivity. From leftto right, (A) the molecules can interact correctly, (B) the secondaryantibody can interact with the target analyte (e.g., in a secondaryepitope), (C) the secondary antibody can interact with the captureprobe, (D) the secondary antibody can interact with the capture probe orother entity through an intermediary molecule, (E) the secondaryantibody can interact with the capture probe without the analyte, and(F) the wrong analyte can be bound by the capture probe and/or thesecondary antibody. These are non-limiting examples. The methods of thedisclosure use an applied electrokinetic force to resolve such issues bypulling apart such non-specific interactions.

FIG. 15 shows an example of performing the method of the presentdisclosure using the apparatus shown in FIG. 14. The various types ofcorrect and non-specifically interacting molecules can be pulled intothe sensing orifice 1500 by an applied electric field 1502. A plot ofsensor output versus applied voltage shows detachment of non-specificbinding 1504, followed by an actual signal 1506 associated with thedisruption of the binding of the correctly bound target analyte.

FIG. 16 shows an example of a process of covalently attaching thecapture probe (e.g., nucleic acids) to the surface. A SiO₂ surface 1600comprising hydroxide moieties 1601 (and/or that is hydroxylated) can besilanized (e.g., using aminosilane 1602). Silanization can be performedin gas phase. A crosslinker (e.g., PMPI 1603) can be used to connect theamine group on the silane and the sulfhydryl group of the 5′thiol-modified primer 1604. The resulting product of the surfacechemistry described herein is shown at 1605.

In practice, sensors can be plasma cleaned, rehydrated andfunctionalized with (3-aminopropyl)-trimethoxysilane (APTMS) using achemical vapor deposition system. The amino-functionalized surfaces canbe subsequently transformed into a thiol-reactive moiety by exposure toa 2.3 mM solution of N-(p-maleimidophenyl) isocyanate (PMPI) inanhydrous toluene at 40° C. for 2 hours under an argon atmosphere. Thesurfaces can be subsequently washed with anhydrous toluene and dried ina stream of argon followed by DNA immobilization using thiolatedoligonucleotides. Prior to immobilization, the thiolated oligos can bereduced using tris(2-carboxyethyl)phosphine (TCEP) as a reducing agentand desalted using a spin column (MWCO=3000). Thiolated oligos can bespotted directly onto sensing chips for 6 hours at 10μM concentration ina 1M NaCl buffer solution under a controlled atmosphere, followed byextensive washing. The various surface modification steps can befollowed by x-ray photoelectron spectroscopy and the presence of theexpected elements and peak shifts confirmed the transformation of thesensing surface. The presence of immobilized nucleic acid can beverified by fluorescence microscopy with appropriately excited SYBR Goldnucleic acids dye. Subsequently, the immobilized oligonucleotides can beused to further increase the pool of possible surface modifications byintroducing the desired probes conjugated to the complementary strand ofthe immobilized nucleic acid. Additional details pertaining tofabrication or operation of the devices described herein can be found inPCT Patent Application Serial No. PCT/US2015/036800, which isincorporated herein in its entirety for all purposes.

FIG. 17 shows an example of an apparatus of the present disclosure,including a fluorescence microscopy image 1701 of TAMRA(5-carboxytetramethylrhodamine) labeled oligonucleotides in an array ofwells for concentrating electric fields, transparent indium tin oxideelectrodes 1702, and a field terminating metal plate of platinumsurrounding the array of wells 1703.

FIG. 18 shows a computer system 1801 that is programmed or otherwiseconfigured to regulate the operation of the apparatus of the presentdisclosure. The computer system 1801 can regulate, for example, flowrates, temperatures, pressures, mechanical manipulations, appliedvoltages or other electrical inputs and/or outputs, and the like.

The computer system 1801 includes a central processing unit (CPU, also“processor” and “computer processor” herein) 1805, which can be a singlecore or multi core processor, or a plurality of processors for parallelprocessing. The computer system 1801 also includes memory or memorylocation 1810 (e.g., random-access memory, read-only memory, flashmemory), electronic storage unit 1815 (e.g., hard disk), communicationinterface 1820 (e.g., network adapter) for communicating with one ormore other systems, and peripheral devices 1825, such as cache, othermemory, data storage and/or electronic display adapters. The memory1810, storage unit 1815, interface 1820 and peripheral devices 1825 arein communication with the CPU 1805 through a communication bus, such asa motherboard. The storage unit 1815 can be a data storage unit (or datarepository) for storing data.

The CPU 1805 can execute a sequence of machine-readable instructions,which can be embodied in a program or software. The instructions may bestored in a memory location, such as the memory 1810. Examples ofoperations performed by the CPU 1805 can include fetch, decode, execute,and writeback.

The storage unit 1815 can store files, such as drivers, libraries andsaved programs. The storage unit 1815 can store programs generated byusers and recorded sessions, as well as output(s) associated with theprograms. The storage unit 1815 can store user data, e.g., userpreferences and user programs. The computer system 1801 in some casescan include one or more additional data storage units that are externalto the computer system 1801, such as located on a remote server that isin communication with the computer system 1801 through an intranet orthe Internet.

The computer system 1801 can be in communication with a system 1830,including a device with integrated fluidics and/or process elements.Such process elements can include sensors, flow regulators (e.g.,valves), and pumping systems that are configured to direct a fluid.

Methods as described herein can be implemented by way of machine (e.g.,computer processor) executable code stored on an electronic storagelocation of the computer system 1801, such as, for example, on thememory 1810 or electronic storage unit 1815. The machine executable ormachine readable code can be provided in the form of software. Duringuse, the code can be executed by the processor 1805. In some cases, thecode can be retrieved from the storage unit 1815 and stored on thememory 1810 for ready access by the processor 1805. In some situations,the electronic storage unit 1815 can be precluded, andmachine-executable instructions are stored on memory 1810.

The code can be pre-compiled and configured for use with a machinehaving a processer adapted to execute the code, or can be compiledduring runtime. The code can be supplied in a programming language thatcan be selected to enable the code to execute in a pre-compiled oras-compiled fashion.

Aspects of the systems and methods provided herein, such as the computersystem 1801, can be embodied in programming. Various aspects of thetechnology may be thought of as “products” or “articles of manufacture”typically in the form of machine (or processor) executable code and/orassociated data that is carried on or embodied in a type of machinereadable medium. Machine-executable code can be stored on an electronicstorage unit, such memory (e.g., read-only memory, random-access memory,flash memory) or a hard disk. “Storage” type media can include any orall of the tangible memory of the computers, processors or the like, orassociated modules thereof, such as various semiconductor memories, tapedrives, disk drives and the like, which may provide non-transitorystorage at any time for the software programming. All or portions of thesoftware may at times be communicated through the Internet or variousother telecommunication networks. Such communications, for example, mayenable loading of the software from one computer or processor intoanother, for example, from a management server or host computer into thecomputer platform of an application server. Thus, another type of mediathat may bear the software elements includes optical, electrical andelectromagnetic waves, such as used across physical interfaces betweenlocal devices, through wired and optical landline networks and overvarious air-links. The physical elements that carry such waves, such aswired or wireless links, optical links or the like, also may beconsidered as media bearing the software. As used herein, unlessrestricted to non-transitory, tangible “storage” media, terms such ascomputer or machine “readable medium” refer to any medium thatparticipates in providing instructions to a processor for execution.

Hence, a machine readable medium, such as computer-executable code, maytake many forms, including but not limited to, a tangible storagemedium, a carrier wave medium or physical transmission medium.Non-volatile storage media include, for example, optical or magneticdisks, such as any of the storage devices in any computer(s) or thelike, such as may be used to implement the databases, etc. shown in thedrawings. Volatile storage media include dynamic memory, such as mainmemory of such a computer platform. Tangible transmission media includecoaxial cables; copper wire and fiber optics, including the wires thatcomprise a bus within a computer system. Carrier-wave transmission mediamay take the form of electric or electromagnetic signals, or acoustic orlight waves such as those generated during radio frequency (RF) andinfrared (IR) data communications. Common forms of computer-readablemedia therefore include for example: a floppy disk, a flexible disk,hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD orDVD-ROM, any other optical medium, punch cards paper tape, any otherphysical storage medium with patterns of holes, a RAM, a ROM, a PROM andEPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wavetransporting data or instructions, cables or links transporting such acarrier wave, or any other medium from which a computer may readprogramming code and/or data. Many of these forms of computer readablemedia may be involved in carrying one or more sequences of one or moreinstructions to a processor for execution.

An aspect of the present disclosure provides an apparatus for detectinga presence of an analyte, comprising: (a) a fluidic chamber adapted tocontain an electrolyte; (b) two or more electrodes capable of generatingan electric field within the fluidic chamber; (c) at least one surfacecomprising an immobilized capture probe capable of binding the analyte,wherein the at least one surface is between the two or more electrodesand in contact with the fluidic chamber, and wherein the at least onesurface comprises an electrically conducting layer and an electricallyinsulating layer; and (d) at least one field confining feature proximalto the at least one surface, which at least one field confining featureis capable of concentrating an electric field surrounding the at leastone field confining feature to a strength of at least about 10³ voltsper meter (V/m). In some cases, the analyte is a charged analyte. Theanalyte can be charged directly, or indirectly by binding to a chargedmolecule such as a charged antibody. The apparatus can further comprisea controller operably coupled to the two or more electrodes, where thecontroller is programmed to apply a voltage across the two or moreelectrodes that is sufficient to generate an applied force on theanalyte.

The at least one field confining feature can be capable of concentratingthe electric field surrounding the at least one field confining featureto any suitable strength. In some embodiments, the field strength is atleast about 10³ volts per meter (V/m), at least about 10⁴ V/m, at leastabout 10⁵ V/m, at least about 10⁶ V/m or at least about 10⁷ V/m. In somecases, the field strength is at most about 10³ V/m, at most about 10⁵V/m, at most about 10⁷ V/m, or at most about 10⁹ V/m.

The voltage applied can sufficient to generate any suitable appliedforce on the charged analyte. In some cases, the applied force is atleast about 1 piconewton (pN), at least about 5 pN, at least about 10pN, at least about 20 pN, at least about 50 pN, at least about 100 pN,at least about 500 pN, at least about 1000 pN or at least about 5000 pNon the analyte. In some instances, the applied force is at most about 1piconewton (pN), at most about 5 pN, at most about 10 pN, at most about20 pN, at most about 50 pN, at most about 100 pN, at most about 500 pN,at most about 1000 pN or at most about 5000 pN on the analyte.

The immobilized capture probe can be capable of binding to the analyteto form a probe-analyte complex on the at least one surface. Theimmobilized capture probe can be capable of binding to the analyte vianon-covalent interaction(s). The non-covalent interaction(s) can bedisrupted through an applied force generated upon the application of avoltage across the two or more electrodes.

In some embodiments, the immobilized capture probe is proximal to the atleast one field confining feature. For example, the capture probe can beimmobilized within about 10 nanometers (nm), within about 20 nm, withinabout 50 nm, within about 100 nm, within about 500 nm, or within about1000 nm of the field confining feature.

The at least one field confining feature can be a plurality of fieldconfining features. For example, the surface can have at least about 5,at least about 10, at least about 50, at least about 100, at least about500, at least about 1000, at least about 5000, at least about 10000, atleast about 50000, at least about 10⁶, or at least about 10⁷ fieldconfinement features.

In some cases, the immobilized capture probe is an antibody. In someinstances, the analyte is an antigen. In some cases, the immobilizedcapture probe is a nucleic acid molecule. In some instances, the analyteis a nucleic acid molecule.

Note that the use of the term “immobilized” does not mean that thecapture probe cannot move. For example, the capture probe can betethered to the surface at one or more locations and move within theelectrolyte due to fluid flow or other forces.

The analyte can have any suitable charge (e.g., in order to provide anadequate applied force). In some embodiments, the analyte has a chargeof at least about 1 e⁻, at least about 10 e⁻, at least about 10² e⁻, atleast about 10³ e⁻, at least about 10⁴ e⁻, at least about 10⁵ e⁻ or atleast about 10⁶ e⁻. In some embodiments, the analyte has a charge of atmost about 1 e⁻, at most about 10 e⁻, at most about 10² e⁻, at mostabout 10³ e⁻, at most about 10⁴ e⁻, at most about 10⁵ e⁻ or at mostabout 10⁶ e⁻. Note that the analyte can have a positive charge or anegative charge. Positive charges of a similar magnitude to thosedisclosed herein are also encompassed by the present disclosure.

The apparatus can further include a controller operably coupled to thetwo or more electrodes, where the controller is programmed to apply apotential difference of less than about 100 volts (V) between the two ormore electrodes. In some cases, the applied voltage is less than about50 V, less than about 10 V, less than about 5 V, or less than about 1V.In some instances, the applied voltage is greater than about 50 V,greater than about 10 V, greater than about 5 V, or greater than about1V.

The at least one surface can provide a wall of the fluidic chamber (asshown in FIG. 1A) and the field confining feature is an orifice in thesurface. In some cases, the field confining feature comprises an orificein the at least one surface. In some cases, the fluidic chambercomprises a top portion and a bottom portion partitioned by the at leastone surface, and wherein the field confining feature is an orifice inthe at least one surface (as shown in FIG. 1B). In some cases, theorifice extends through the surface. The orifice can be, withoutlimitation, an indentation, a well, a pore, a channel, a gap, or a slit.The field confining feature can be any shape, including circular, oval,square, rectangular, or any polygon.

The field confining feature can have any suitable diameter. In somecases, the diameter is less than about 50 micrometers, less than about10 micrometers, less than about 5 micrometers, less than about 1micrometer, less than about 500 nanometers, less than about 100nanometers, or less than about 50 nanometers. In some cases, thediameter is greater than about 50 micrometers, greater than about 10micrometers, greater than about 5 micrometers, greater than about 1micrometer, greater than about 500 nanometers, greater than about 100nanometers, or greater than about 50 nanometers.

The field confining feature can have any suitable aspect ratio. In somecases, the aspect ratio is the ratio of the longest dimension of thefield confining feature to the shortest dimension of the field confiningfeature. In some instances, the aspect ratio is the ratio of the widthof the field confining feature to the depth of the field confiningfeature. Without limitation, the field confining feature can have anaspect ratio of at least about 0.1, at least about 0.5, at least about1, at least about 2, at least about 3, at least about 5, at least about10, at least about 50, at least about 100, or more. The field confiningfeature can have a sharp edge (e.g., having an angle of at least about60 degrees). The field confining feature can be an elevated portion ofthe at least one surface.

The electrically conducting layer can be in communication with theelectrolyte, such as physically contacting or in electricalcommunication. The electrically conducting layer can be electricallyisolated from the electrolyte, and wherein, during use, the electricallyconducting layer is electrically biased. The electrically conductinglayer can comprise any suitable metal, such as platinum, copper, gold orsilver.

The apparatus can further comprise a sensing electrode configured tomeasure a charge signal associated with the presence of the analyte. Thesensing electrode can be proximal to the at least one field confiningfeature (e.g., within about 10 nanometers (nm), within about 20 nm,within about 50 nm, within about 100 nm, within about 500 nm, or withinabout 1000 nm of the field confining feature).

The apparatus can further comprise a light source and a detectorconfigured to detect an optical signal associated with the presence ofthe analyte. The optical signal can be a fluorescence signal. In somecases, the fluorescence signal is a fluorescence resonance energytransfer, FRET, signal. The fluorescence signal can be provided by afluorescent probe covalently or non-covalently attached to the analyte.

The apparatus can further comprise a detector configured to detect asurface plasmon resonance signal associated with the presence of theanalyte.

The at least one surface can be substantially planar. The at least onesurface can be part of (or comprise) an array of sensors, wherein eachsensor of the array comprises at least one field confining feature and aplurality of immobilized capture probes. The plurality of immobilizedcapture probes of a given sensor of the array can be proximal to thefield confining feature for the given sensor of the array. The pluralityof immobilized capture probes of a given sensor of the array can beclonal. In some cases, a given sensor of the array comprises a pluralityof immobilized capture probes that is unique relative to another sensorof the array.

The distance between a given sensor of the array and a nearest neighborsensor can be any suitable distance. In some cases, the distance is atleast about 50 nanometers (nm), at least about 100 nm, at least about150 nm, at least about 200 nm, at least about 250 nm, at least about 500nm, at least about 1000 nm, at least about 5000 nm or at least about10000 nm. In some cases, the distance is at most about 50 nanometers(nm), at most about 100 nm, at most about 150 nm, at most about 200 nm,at most about 250 nm, at most about 500 nm, at most about 1000 nm, atmost about 5000 nm or at most about 10000 nm.

The array can comprise any suitable number of sensors. In some cases,the array comprises at least about 5, at least about 10, at least about50, at least about 100, at least about 500, at least about 1000, atleast about 5000, at least about 10000, at least about 50000, at leastabout 10⁶, or at least about 10⁷ sensors.

In another aspect, the disclosure provides method for detecting apresence of a target analyte, comprising (a) activating at least onesensor comprising one or more surfaces between two or more electrodes,wherein the one or more surfaces comprise two or more immobilizedcapture probes; (b) bringing the one or more surfaces in contact with asolution containing or suspected of containing a target analyte and anon-target analyte, under conditions that are sufficient to permit thetarget analyte and non-target analyte to each bind to a given one of thetwo or more immobilized capture probes; (c) applying a voltage acrossthe two or more electrodes that is sufficient to release the non-targetanalyte, but not the target analyte, from a given one of the two or moreimmobilized capture probes; and (d) detecting a presence of the targetanalyte on the one or more surfaces. The target analyte and/or thenon-target analyte can be a charged analyte. The voltage can besufficient to exert an applied force of at least about 1 piconewton (pN)on the non-target analyte. Operations (c) and/or (d) can be performed inthe absence of washing the one or more surfaces.

The method of can further comprise applying an additional voltage acrossthe two or more electrodes that is sufficient to release the targetanalyte from a given one of the two or more immobilized capture probes.The additional voltage can be sufficient to exert an applied force of atleast about 1 piconewton (pN) on the target analyte.

In various embodiments, the force applied on the target or non-targetanalytes is at least about 1 piconewton (pN), at least about 5 pN, atleast about 10 pN, at least about 20 pN, at least about 50 pN, at leastabout 100 pN, at least about 500 pN, at least about 1000 pN or at leastabout 5000 pN. In some instances, the applied force is at most about 1piconewton (pN), at most about 5 pN, at most about 10 pN, at most about20 pN, at most about 50 pN, at most about 100 pN, at most about 500 pN,at most about 1000 pN or at most about 5000 pN on the analyte.

The method can further comprise detecting the presence of the targetanalyte on the one or more surfaces subsequent to application of theadditional voltage. The voltage can be less than the additional voltage.

The voltage and the additional voltage can be individual voltages of acontinuously applied voltage that is changed over time, as shown in FIG.6A or FIG. 8A.

The voltage and/or the additional voltage can be applied in the absenceof washing the one or more surfaces. For example, the methods of thepresent disclosure can be performed in complex mixtures such asbiological fluids (e.g., blood) or environmental samples.

The applied voltage can be sufficient to generate an electric fieldhaving a strength of less than about 10⁹ volts per meter (V/m) and theadditional applied voltage can be sufficient to generate an additionalelectric field having a strength of less than about 10⁹ V/m, less thanabout 10⁸ V/m, less than about 10⁷ V/m, less than about 10⁶ V/m, lessthan about 10⁵ V/m, less than about 10⁴ V/m, less than about 10³ V/m, orlower.

In some cases, each of the target analyte and non-target analytenon-covalently binds to a given one of the two or more immobilizedcapture probes to form a target probe-analyte complex immobilized and anon-target probe-analyte complex immobilized on the one or moresurfaces, respectively.

The applied voltage can be increased from a first voltage to a secondvoltage over time. The applied voltage can be increased from the firstvoltage to the second voltage at a rate of at least about 1 millivoltper second (mV/s), at least about 5 mV/s, at least about 10 mV/s, atleast about 50 mV/s, at least about 100 mV/s, at least about 500 mV/s,at least about 1000 mV/s, at least about 5000 mV/s, or more.

In some cases, the applied voltage is increased from the voltage to theadditional voltage over a period of time greater than about 10microseconds, greater than about 50 microseconds, greater than about 100microseconds, greater than about 500 microseconds, greater than about1000 microseconds, greater than about 5000 microseconds, greater thanabout 10000 microseconds or more. In some cases, the applied voltage isincreased from the voltage to the additional voltage over a period oftime less than about 1 second.

The applied voltage can be a periodic voltage waveform. The periodicvoltage waveform can have any suitable frequency, such as a frequency ofless than about 10⁹ Hertz (Hz), less than about 10⁸ Hz, less than about10⁷ Hz, less than about 10⁶ Hz, less than about 10⁵ Hz, less than about10⁴ Hz, less than about 10³ Hz, less than about 10² Hz, less than about10 Hz, less than about 1 Hz, less than about 10⁻² Hz, or less than about10⁻³ Hz. The periodic voltage waveform can have a frequency of greaterthan about 10⁹ Hertz (Hz), greater than about 10⁸ Hz, greater than about10⁷ Hz, greater than about 10⁶ Hz, greater than about 10⁵ Hz, greaterthan about 10⁴ Hz, greater than about 10³ Hz, greater than about 10² Hz,greater than about 10 Hz, greater than about 1 Hz, greater than about10⁻² Hz, or greater than about 10⁻³ Hz.

The method of can further comprise monitoring a signal indicative of thepresence of the target analyte on the one or more surfaces as theapplied voltage is varied over time.

The method can further comprise determining a binding force of thetarget analyte bound to a given one of the two or more immobilizedcapture probes (e.g., the force required to dissociate the analyte fromthe binding probe).

The applied force can be less than a binding force of the target analytebound to a given one of the two or more immobilized capture probes.

The electrolyte (solution) can comprise charged molecules that bind withthe target analyte and/or the non-target analyte, to provide a change incharge on the target analyte and/or non-target analyte. The chargedmolecules can be antibodies.

The presence of the target analyte on the one or more surfaces can bedetected by measuring a signal associated with the target analyte. Thesignal can be a charge signal. The charge signal can be detected using asensing electrode and a reference electrode. The charge signal can be avoltage.

The presence of the target analyte on the one or more surfaces can bedetected by measuring an optical signal associated with the targetanalyte. The optical signal can be a fluorescence signal. Thefluorescence signal can be provided by a fluorescent probe covalently ornon-covalently attached to the target analyte.

The presence of the target analyte on the one or more surfaces can bedetected using surface plasmon resonance.

The one or more surfaces can comprise a membrane having a thickness ofbetween about 0.08 nanometers and 1 millimeter. The one or more surfacescan include a plurality of surfaces. Each of the plurality of surfacescan comprise a subset of the two or more immobilized capture probes.

The voltage can be a direct current voltage or an alternating currentvoltage.

In another aspect, the present disclosure provides a system fordetecting a presence of a target analyte, comprising: (a) at least onesensor comprising one or more surfaces between two or more electrodes,wherein the one or more surfaces comprise two or more immobilizedcapture probes; (b) a solution chamber in fluid communication with theone or more surfaces, wherein the solution chamber is configured toretain a solution containing or suspected of containing the targetanalyte and a non-target analyte, under conditions that are sufficientto permit the target analyte and non-target analyte to each bind to agiven one of the two or more immobilized capture probes; and (c) acontroller that is operably coupled to the two or more electrodes,wherein the controller is programmed to (i) apply a voltage across thetwo or more electrodes that is sufficient to release the non-targetanalyte, but not the target analyte, from a given one of the two or moreimmobilized capture probes, and (ii) detect a presence of the targetanalyte on the one or more surfaces.

The target analyte and/or the non-target analyte can be a chargedanalyte. In some cases, the target analyte and/or the non-target analytecan have a first charge (e.g., zero charge) and may obtain a secondcharge (e.g., +2, −2) upon coupling to a charged molecule.

The voltage across the two or more electrodes can be sufficient to exertan applied force of at least 1 piconewton on the non-target analyte. Theat least one sensor can comprise an array of sensors. Each sensor of thearray of sensors can be independently addressable (e.g., havemeasurements taken from it).

It should be understood from the foregoing that, while particularimplementations have been illustrated and described, variousmodifications can be made thereto and are contemplated herein. Forexample, the embodiments described herein can be combined with ormodified to yield yet more embodiments of the present invention. It isalso not intended that the invention be limited by the specific examplesprovided within the specification. While the invention has beendescribed with reference to the aforementioned specification, thedescriptions and illustrations of the preferable embodiments herein arenot meant to be construed in a limiting sense. Furthermore, it shall beunderstood that all aspects of the invention are not limited to thespecific depictions, configurations or relative proportions set forthherein which depend upon a variety of conditions and variables. Variousmodifications in form and detail of the embodiments of the inventionwill be apparent to a person skilled in the art. It is thereforecontemplated that the invention shall also cover any such modifications,variations and equivalents. It is intended that the following claimsdefine the scope of the invention and that methods and structures withinthe scope of these claims and their equivalents be covered thereby.

1-57. (canceled)
 58. An apparatus for detecting a presence of ananalyte, comprising: a fluidic chamber adapted to contain anelectrolyte; two or more electrodes capable of generating an electricfield within the fluidic chamber; at least one surface comprising animmobilized capture probe capable of binding the analyte, wherein the atleast one surface is between the two or more electrodes and in contactwith the fluidic chamber, and wherein the at least one surface comprisesan electrically conducting layer and an electrically insulating layer;and at least one field confining feature proximal to the at least onesurface, which at least one field confining feature has a diametergreater than about 1 micrometer (μm) and is capable of concentrating anelectric field surrounding the at least one field confining feature to astrength of at least about 10³ volts per meter (V/m).
 59. The apparatusof claim 58, wherein the analyte is a charged analyte.
 60. The apparatusof claim 58, wherein the at least one field confining feature is capableof concentrating the electric field surrounding the at least one fieldconfining feature to a strength of less than about 10⁵ V/m.
 61. Theapparatus of claim 58, further comprising a controller operably coupledto the two or more electrodes, wherein the controller is programmed toapply a voltage across the two or more electrodes that is sufficient togenerate an applied force on the analyte.
 62. (canceled)
 63. Theapparatus of claim 61, wherein the voltage is sufficient to generate anapplied force of at least about 20 pN on the analyte.
 64. The apparatusof claim 58, wherein the immobilized capture probe is capable of bindingto the analyte to form a probe-analyte complex on the at least onesurface.
 65. The apparatus of claim 64, wherein the immobilized captureprobe is capable of binding to the analyte via non-covalentinteraction(s).
 66. The apparatus of claim 65, wherein the non-covalentinteraction(s) are disruptable through an applied force generated uponthe application of a voltage across the two or more electrodes.
 67. Theapparatus of claim 64, wherein the immobilized capture probe is proximalto the at least one field confining feature.
 68. (canceled)
 69. Theapparatus of claim 58, wherein the immobilized capture probe is anantibody.
 70. (canceled)
 71. The apparatus of claim 58, wherein theimmobilized capture probe is a nucleic acid molecule. 72-74. (canceled)75. The apparatus of claim 58, further comprising a controller operablycoupled to the two or more electrodes, wherein the controller isprogrammed to apply a potential difference of less than about 100 volts(V) between the two or more electrodes.
 76. The apparatus of claim 58,wherein the at least one surface provides a wall of the fluidic chamberand the field confining features is an orifice in the surface.
 77. Theapparatus of claim 58, wherein the field confining feature is an orificein the at least one surface. 78-79. (canceled)
 80. The apparatus ofclaim 77, wherein the orifice is an indentation, a well, a pore, achannel, a gap, or a slit. 81-87. (canceled)
 88. The apparatus of claim58, wherein the electrically conducting layer is in communication withthe electrolyte. 89-90. (canceled)
 91. The apparatus of claim 58,further comprising a sensing electrode configured to measure a chargesignal associated with the presence of the analyte.
 92. The apparatus ofclaim 91, wherein the sensing electrode is proximal to the at least onefield confining feature. 93-97. (canceled)
 98. The apparatus of claim58, wherein the at least one surface is part of an array of sensors,wherein each sensor of the array comprises a field confining feature anda plurality of immobilized capture probes. 99-109. (canceled)
 110. Theapparatus of claim 58, wherein the diameter is greater than about 5 μm.